Corneal nerve loss in patients with TIA and acute ischemic stroke in relation to circulating markers of inflammation and vascular integrity

Vascular and inflammatory mechanisms are implicated in the development of cerebrovascular disease and corneal nerve loss occurs in patients with transient ischemic attack (TIA) and acute ischemic stroke (AIS). We have assessed whether serum markers of inflammation and vascular integrity are associated with the severity of corneal nerve loss in patients with TIA and AIS. Corneal confocal microscopy (CCM) was performed to quantify corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD) and corneal nerve fiber length (CNFL) in 105 patients with TIA (n = 24) or AIS (n = 81) and age matched control subjects (n = 56). Circulating levels of IL-6, MMP-2, MMP-9, E-Selectin, P-Selectin and VEGF were quantified in patients within 48 h of presentation with a TIA or AIS. CNFL (P = 0.000, P = 0.000), CNFD (P = 0.122, P = 0.000) and CNBD (P = 0.002, P = 0.000) were reduced in patients with TIA and AIS compared to controls, respectively with no difference between patients with AIS and TIA. The NIHSS Score (P = 0.000), IL-6 (P = 0.011) and E-Selectin (P = 0.032) were higher in patients with AIS compared to TIA with no difference in MMP-2 (P = 0.636), MMP-9 (P = 0.098), P-Selectin (P = 0.395) and VEGF (P = 0.831). CNFL (r = 0.218, P = 0.026) and CNFD (r = 0.230, P = 0.019) correlated with IL-6 and multiple regression analysis showed a positive association of CNFL and CNFD with IL-6 (P = 0.041, P = 0.043). Patients with TIA and AIS have evidence of corneal nerve loss and elevated IL6 and E-selectin levels. Larger longitudinal studies are required to determine the association between inflammatory and vascular markers and corneal nerve fiber loss in patients with cerebrovascular disease.

Whilst elevated E-selectin levels were independently associated with poorer outcomes in patients with acute ischemic stroke 14 , a recent prospective analysis of the EPIC-Heidelberg study showed no association between E-selectin or P-selectin levels and the risk of incident stroke 15 . These observations suggest a complex temporal relationship between changing levels of circulating markers of inflammation and vascular integrity with outcomes in acute ischemic stroke.
Corneal confocal microscopy (CCM) is a non-invasive ophthalmic imaging technique that has identified corneal nerve loss in patients with diabetic neuropathy [16][17][18] , other peripheral neuropathies 19 , multiple sclerosis 20,21 and dementia 22 . Recently, we have shown corneal nerve loss in patients with TIA 23 , acute ischemic stroke [24][25][26][27] and recurrent stroke 28 . Increased plasma adhesion molecules including P-selectin and ICAM-1 predict the development of diabetic neuropathy 29 and elevated IL-6 has been associated with distal neuropathy in subjects with impaired glucose tolerance 30 .
We have assessed the association between corneal nerve loss and circulating vascular and inflammatory markers in patients with TIA and stroke.

Results
Clinical, metabolic and corneal confocal microscopy measures. Clinical, metabolic and CCM parameters in the study participants are given in Table 1.

Multiple linear regression.
Multiple regression analysis showed a positive association between CNFL (Table 3) and CNFD (Table 4) with IL-6 and a negative association with age. The CNBD distribution was skewed and was not included in the regression analysis.

Discussion
In the present study, we show that patients admitted with TIA or acute ischemic stroke have corneal nerve loss. These data support our previous smaller studies showing a loss of corneal nerves in patients with TIA 23 and acute ischemic stroke 24 . Moreover, we now show that the severity of corneal nerve loss is comparable between patients with TIA and major stroke. Recently, we have shown greater corneal nerve loss in patients with recurrent stroke, despite comparable vascular risk factors suggesting that assessment of corneal nerve integrity may be a more robust measure of risk of stroke and recurrent stroke compared to the evaluation of conventional risk factors for stroke 28 . Recent skin biopsy studies have also shown a loss of intraepidermal nerve fibres in patients with stroke,   www.nature.com/scientificreports/ particularly those with central post stroke pain 31,32 . Given that we have previously shown comparable reductions in intraepidermal nerves and corneal nerves in diabetic neuropathy 33 , our observations suggest that our finding of corneal nerve loss in TIA and stroke represents more global evidence of nerve loss in stroke and TIA. Vascular factors play an integral role in the development and progression of ischemic stroke. Mounting evidence shows that circulating biomarkers are associated with incident stroke 7 and poorer outcomes after stroke 5,6,12 . Endothelial extracellular vesicles have prothrombotic properties and we have recently shown an increase in patients with TIA and ischemic stroke 34 . The current study did not find an elevation in E-selectin, P-selectin or VEGF or a relationship with corneal nerve loss in patients with TIA or stroke. Although, previously, we have shown that corneal nerve fibre loss is independently associated with the presence of white matter hyperintensities in patients with ischemic stroke 26 . We have also shown a reduction in corneal nerves and endothelial cell density in patients with TIA and minor ischemic stroke 23 and an independent association between corneal endothelial cell density, area and perimeter and acute ischemic stroke 25 .
In the present study, we show that patients with AIS have greater IL-6 and MMP-2 levels compared to TIA which may reflect the severity of neurodegeneration in AIS. The level of IL-6 at admission has been associated with the severity of infarct and neurological outcomes on day 28 35 . Furthermore, the level of IL-6, fibrinogen and white blood cells had a comparable predictive ability to admission NIHSS and infarct size on recovery from stroke at 6-months 36 . It is important to note that the time course for appearance of various biomarkers may vary. Thus, monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) appear within hours of a stroke, but tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), C-reactive protein (CRP), and S100B appear after 12-24 h and each biomarker had differing abilities to predict 90-day outcomes of stroke 37 . More recently, IL-6 levels correlated with the severity of stroke at admission based on the NIHSS and mRS and also predicted recurrence of stroke 38 . The Reasons for Geographic and Racial Differences in Stroke (REGARDS) study enrolled 30,237 participants and showed that IL-6, but not IL-8 or IL-10 was strongly associated with risk of incident stroke over 5.4 years 39 .
IL-6 has been traditionally considered to be a pro-inflammatory cytokine integral to initiating the acute phase response of the immune system 40,41 . However, it is increasingly recognized as a multifunctional cytokine capable of eliciting both pro-and anti-inflammatory effects 42 . Indeed, in the present study, higher levels of IL-6 were independently associated with a higher CNFD and CNFL, despite a loss of corneal nerves in patients with TIA and stroke. In this context, IL-6 has been associated with axonal regeneration following inflammation 43 and intrathecal delivery of IL-6 leads to activation of pyramidal cells in the sensory motor cortex following spinal cord injury 44 . In the context of peripheral nerves, IL-6 receptors are expressed on Schwann cells and following nerve transection the administration of IL-6/IL-6R fusion protein resulted in a four-fold increase in myelinated nerve fiber regrowth 45 . IL-6 is also intimately involved in axonal regeneration as it induces increased expression of growth associated protein-43 (GAP-43) 46 . IL-6 deficient mice display reduced amplitude of sensory action potentials and temperature sensitivity and impaired axonal regeneration following sciatic nerve crush injury 47 .
This study has limitations. CCM was not performed in patients with severe disability due to their inability to cooperate during the CCM procedure. This may have biased the outcomes as the results may have been even more pronounced in those with more severe stroke. Second, circulatory markers and neurological outcomes were only measured at one time point.
In conclusion, we show greater corneal nerve loss in patients with stroke and TIA compared to healthy controls and contrary to our initial hypothesis we show that elevated IL-6 levels were independently associated with greater corneal nerve measures. However, to establish a mechanistic link between circulating markers of inflammation and vascular integrity in relation to corneal nerve integrity and outcomes in TIA and stroke, longitudinal studies are required.

Materials and methods
One hundred and five patients-admitted with AIS (n = 24) and acute ischemic stroke (n = 81) were recruited from the stroke ward at Hamad General Hospital. The diagnosis of TIA and stroke were confirmed clinically and radiologically using AHA criteria 48 . Exclusion criteria included patients with a known history of ocular trauma or surgery, glaucoma, dry eye and corneal dystrophy 49 . Demographic (age, gender, ethnicity) and clinical (blood pressure, HbA 1c , lipid profile) data were obtained from the admission patients' electronic health records. All patients underwent assessment of the National Institutes of Health Stroke Scale (NIHSS) at presentation. This study adhered to the tenets of the declaration of Helsinki and was approved by the Institutional Review Board of Weill Cornell Medicine (15-00021) and Hamad General Hospital (15304/15). Informed, written consent was obtained from all patients/guardians before participation in the study. Statistical analysis. Statistical justification for the number of participants was based on a power analysis using the freeware program G*Power version 3.0.10 for α (type 1 error) of 0.05 and power (1 − type 2 error) of 0.80 using corneal nerve fibre density mean (37.12 vs 29.18) and standard deviation (8.35 and 7.16) in healthy controls and patients with stroke from our previous study 24 which established that we needed a minimum of 64 patients with stroke. All statistical analyses were performed using IBM SPSS Statistics software Version 25. Normality of the data was assessed using the Shapiro-Wilk test and by visual inspection of the histogram and a normal Q-Q plot. Data are expressed as mean ± standard deviation (SD). Mann Whitney test (for non-normally distributed variables) and t-test (for normally distributed variables) were performed to find the differences between two groups, and one-way ANOVA was performed to find the differences between three groups and Bonferroni correction was performed. Pearson correlation was performed to find association between corneal nerves and circulatory biomarkers. Multiple linear regression analysis was conducted to evaluate the independent association between CNFL and CNFD and covariates.
Ethics approval. This study adhered to the tenets of the declaration of Helsinki and was approved by the Institutional Review Board of Weill Cornell Medicine (15-00021) and Hamad General Hospital (15304/15).
Consent to participate. Informed, written consent was obtained from all patients/guardians before participation in the study.

Consent for publication.
Written consent was obtained from all patients/guardians for publications.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.